Posttranslational modifications (PTMs) of histones play fundamental roles in diverse biological processes such as transcriptional regulation and genome maintenance, and aberrant regulations of histone PTMs have been increasingly implicated in many diseases including cancer. Basic research on well-studied histone lysine acetylation and methylation has led to the development of epigenetic therapies that are transitioning to the clinic, especially for treating hematological malignancies. In contrast, littl is known about an expanding group of newly discovered histone PTMs that include crotonylation, propionylation, butyrylation, malonylation succinylation, and 2-hydroxyisobutyrylation all of which are chemically related to histone acetylation and collectively termed lysine acylations. The existence of such diverse histone acylations in addition to Kac suggests that differential regulation of these PTMs has broad and important biological roles yet to be discovered. Focusing on histone lysine crotonylation, our recently published and preliminary studies support a role of histone crotonylation in transcriptional activation and reveal its potential link to oncogenesis through its interaction with cancer-related proteins. The general objective of our proposed research is to understand the fundamental role and mechanism of histone lysine crotonylation in the regulation of transcription in physiological and pathological conditions. To reach this goal we propose a multi-disciplinary approach where we will combine expertise from three labs to tackle this project. Through approaches in chromatin biology (Allis Lab, Aim 1), we will identify and characterize (a) the factors responsible for the enzymatic and metabolic regulation of histone Kcr and (b) the proteins that bind with Kcr and mediate its downstream effect. Through the lens of transcription (Roeder Lab, Aim 2) we will determine the direct contribution of histone lysine crotonylation and its associated proteins to gene activation and define the underlying mechanism of action using biochemically defined systems with reconstituted normal and disease-associated factors. Finally, by focusing on the role of Kcr in leukemias (Armstrong Lab, Aim3) we will define the genome-wide consequences histone lysine crotonylation has on chromatin landscape and gene expression in normal and transformed cells, and examine the functional importance of these modifications in leukemias via cell based assays and mouse models. Through this integrated approach we will work towards identifying and characterizing new proteins and pathways involved in normal and pathogenic gene regulation with the potential to uncover novel targets for epigenetic therapy of cancer.